articles The structure of DNA in the nucleosome core Timothy J. Richmond & Curt A. Davey ETH Zu¨rich, Institut fu¨r Molekularbiologie und Biophysik, ETH-Ho¨nggerberg, CH-8093 Zu¨rich The authors contributed equally to this work ........................................................................................................................................................................................................................... The 1.9-A˚ -resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non- histone protein–DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fibre formation and chromatin remodelling. DNA in eukaryotic cells is packaged repetitively into nucleosomes mobility15. Their mechanism of action most probably derives from by means of extensive association with histone proteins1–3. The the innate ability of nucleosomes to ‘slide’ along DNA without hierarchical chromatin structure formed is the genomic substrate releasing it3. relevant to the vital processes of DNA replication, recombination, An accurate, atomic-level description of DNA conformation in transcription, repair and chromosome segregation, and to the the nucleosome core and comparison with naked DNA is essential pathological progression of cancer and viral disease. Although to an understanding of chromatin properties such as those resulting nucleosomal organization of DNA is essentially ubiquitous from nucleosome position and mobility. Previously, the probable throughout genomes and generally repressive to gene expression, sequence-dependent features of nucleosomal DNA were proposed it also contributes to gene transcription in a gene-specific manner, by extrapolation from high-resolution DNA oligonucleotide struc- suggesting that nucleosome positioning in gene promoter regions is tures16. Here, a direct analysis of nucleosome DNA is made, important for genuine gene regulation in vivo4–6. The question permitted by the high quality of the DNA structure in the 1.9-A˚ - therefore arises of how chromatin structure, in which DNA is resolution X-ray structure of the nucleosome core particle normally highly compacted, permits site-specific access to regula- (NCP147)17,18, comparable to high-resolution oligonucleotide tory factors and more extensive exposure to the transcription structures. The persistently tight curvature of nucleosome core apparatus. DNA causes it to take on conformations not obviously present in The answer is likely to require a knowledge of DNA conformation oligonucleotides but about which there has been speculation for in the nucleosome core. The core comprises 147 base pairs (bp) of many years19. Furthermore, comparison of the DNA twist values DNA and the histone octamer; compared with the nucleosome, it measured for the 147-bp and 146-bp structures with the DNA lacks only 10–90 bp of linker DNA envisaged to be naked or bound sequence periodicity found in bulk chromatin indicates that the to histone H1. The histone-fold domains of the octamer organize DNA stretching observed in the nucleosome core is commonplace the central 129 of 147 bp in 1.59 left-handed superhelical turns with in vivo. a diameter only fourfold that of the double helix. The relatively straight 9-bp terminal segments contribute little to the curvature of Excess DNA curvature the complete 1.67-turn superhelix7. So far, the site-specific regulat- The ideal DNA superhelix that best fits the NCP147 DNA was ory factors that have been discovered bind the linker or terminal constructed from uniformly distributed base pairs by using B-form regions of the intact nucleosome. The lack of binding to the central DNA geometry. It has a radius of 41.9 A˚ and a pitch of 25.9 A˚ region of the superhelix might simply be a consequence of bending (Fig. 1a). Each base-pair step comprising two adjacent base pairs the double helix or, additionally, of unusual DNA conformations contributes 4.538 to the curvature of the ideal superhelix. However, induced by histone binding. At least one protein, HIV-1 integrase, the NCP147 superhelix is not bent uniformly owing to (1) the does prefer DNA bent around the nucleosome in contrast to naked anisotropic flexibility of DNA, (2) local structural features intrinsic DNA8. to the DNA sequence, and (3) irregularities dictated by the under- The initiation of DNA-dependent nuclear processes in the con- lying histone octamer. The NCP147 DNA is generally B-form as text of chromatin implies that nucleosome position is biased by the judged by the criterion of the phosphate ‘Z-coordinate’ with a value DNA sequence to facilitate access by initiation factors. Numerous of 20.18 ^ 0.69 A˚ (mean ^ s.d.; range 22.32 to 1.45 A˚ ). On the examples of positioned nucleosomes in gene promoter regions have basis of oligonucleotide structures, values falling in the range from been described both in vivo and in vitro9–11. Preferential positioning 21 to 0 are indicative of the B-form, whereas values above 2 signify could place factor-binding sequences in nucleosome linker or the A-form20. Other standard conformational parameters also terminal region DNA. Furthermore, nucleosomes are intrinsically indicate that the DNA is predominantly B-form (see Supplementary mobile and yield access to their DNA in vitro, allowing even RNA Table 1). polymerase to transcribe nucleosomal DNA without causing dis- Remarkably, the NCP147 DNA has double the base-pair-step sociation of the histone octamer12–14. In vivo, energy-dependent curvature necessary to produce the DNA superhelix path. The ideal chromatin remodelling factors, targeted by gene regulatory proteins superhelix fit to the NCP147 DNA yields 1.67 superhelical turns for and acting directly on the nucleosome core, augment nucleosome 133.6 bp (1.84 superhelical turns for 147 bp), bending the DNA NATURE | VOL 423 | 8 MAY 2003 | www.nature.com/nature © 2003 Nature Publishing Group 145 articles through 600.08, whereas the actual curvature of NCP147 DNA is for the ideal and actual curvatures, respectively, and thus the 1,333.38 (both scalar curvature values were calculated with the terminal regions do not account for the discrepancy. The excess is program Curves21). The use of only the central 129 bp to remove not adequately explained by the zigzag path of the NCP147 super- the effect of straightening of the superhelical path at the termini helix, as the overall root-mean-square deviation from the ideal path yields values of 579.38 and 1248.68 (9.75 ^ 5.138 per base-pair step) is only 1.42 A˚ (Fig. 1a). We find that the greater part of the excess curvature is a consequence of alternation of DNA form not revealed by criteria based on oligonucleotide structures. Determining the component of curvature directed towards superhelix formation exposes the origin of the form changes. The potential contributions to curvature from the roll and tilt (see Supplementary Fig. 1) of each base-pair step vary as sinusoidal functions of the accumulated twist angle (V) along the length of the double helix. To evaluate the actual contributions, the roll and tilt values for each base-pair step are multiplied by the corresponding cos V (CAT) and sin V (SAT), respectively. A convenient origin for the summation of V is taken as the central base pair located on the pseudo-two-fold symmetry axis of the particle (base pair 0 or dyad position). For the ideal superhelix, the CAT and SAT values multiplied by 4.538 yield the roll and tilt contributions to superhelix curvature at each base-pair step (iCATand iSAT; Fig. 1b). The actual roll and tilt values for NCP147 DNA are moderately well correlated with CAT (R ¼ 0.72, P , 0.0001) and SAT (R ¼ 0.54, P , 0.0001), respectively. The sum of roll and tilt multiplied by CAT and SAT, respectively, over all base-pair steps yields a curvature free of the effect of path deviations between the ideal and actual super- helices. The resulting value is 897.08, or 1.5-fold that required to accommodate the full NCP147 superhelix. Unlike the computer-generated ideal superhelix, for which cur- vature derives equally from roll and tilt (iCAT and iSAT; Fig. 1b), curvature for oligonucleotide and NCP147 DNA stems predomi- nately from the roll parameter. For DNA oligonucleotide structures, roll is favoured over tilt by 2:1 (ref. 22), presumably because it requires less base-pair unstacking for any particular bend angle. In the nucleosome core, this ratio is virtually identical at 1.9:1, but when the components that contribute to the superhelix only are considered, the ratio is 3.0:1. Curvature into the superhelix is therefore accumulated every approximately five base pairs, where either the major or minor groove faces the histone octamer. Never- theless, the NCP147 tilt component contributes 77% of that for the ideal superhelix (Fig. 1b; compare tilt and iSAT plots). In contrast, the roll component is 222% of the ideal contribution (Fig. 1b; compare roll and iCAT plots). Analyses of crystal structures of DNA oligonucleotides and protein–DNA complexes normally equate DNA bending with DNA curvature23 and rely on base-pair-step parameters to explain bending behaviour; for example, by using roll and tilt22,24, roll and twist16,25,26 or roll, tilt, twist, slide, shift and rise24,27. This type of approach is inadequate for nucleosome core DNA, given the substantial excess curvature that contributes to superhelix for- mation.